The long-term objective of this research program is to elucidate the function of human cytomegalovirus (HCMV) genes that regulate the interaction of the virus with its host cell and thereby control the processes of viral replication and pathogenesis. This proposal will study HCMV proteins that are delivered to the cell as constituents of the virion. These proteins have the potential to exert profound effects on the virus-host interaction, because they are present at the very start of the infectious process. Our studies will be performed in fibroblasts, the standard host cell used for HCMV studies in the laboratory, and in epithelial cells, a cell type that is central to HCMV replication and spread in infected people. We will investigate both laboratory strains and clinical isolates of HCMV. Our technical approach will combine genetics, molecular biology and proteomics. Our specific aims focus on the functional analysis of three virus-coded virion proteins: pUL83, pUS22 and pUL23/pUL24. pUL83 is the most abundant virion constituent. We and others have shown that it inhibits the induction of cellular anti-viral genes at the start of infection, and we have discovered that it interacts with the cellular IFI-16 protein. We propose to investigate the mechanism by which pUL83 blocks the protective cellular response and how its interaction with IFI- 16 contributes to its function. pUS22 has no known effect on HCMV replication in fibroblasts. We now have discovered that it is required for efficient replication in epithelial cells, and we have shown that it binds to the cellular SF2/ASF protein, a multi-functional splicing factor. We will investigate the host range function of pUS22 in epithelial cells, and we will explore the consequences of its interaction with SF2/ASF. Since depletion of SAF2/ASF and HCMV infection are both known to induce genomic instability, we will test the hypothesis that pUS22 blocks SF2/ASF function and thereby induces genomic instability. We have shown that the pUL23 and pUL24 proteins form a complex and that this complex functions after the viral genome reaches the nucleus and before immediate-early mRNAs function. We will use mutant viruses to further delineate the very early point in the replication cycle at which this protein complex functions, we will identify cellular proteins with which the complex interacts, and we will explore the functional consequences of these interactions.